March, 1940
PFSPARATION AND
[CONTRIBUTION FROM THE
DEGRADATION OF LITHOCHOLIC ACID
569
DIVISION OF BIOCHEMISTRY, THEMAYOFOUNDATION, ROCHESTER, MINNESOTA]
The Preparation and Degradation of Lithocholic Acid BY WILLARD M. HOE" The importance of desoxycorticosterone as the most active of the crystalline substances thus far isolated from the adrenal gland makes it desirable to develop methods for its preparation. Stigmasterol' and cholesterolZ have been used as raw materials for its preparation by several procedures. It is the purpose of this note to describe some improved procedures for the conversion of
AND
HAROLD L. MASON
have described similar degradations. The melting points and specific rotations of the compounds so obtained are given in Table I since some of these values are not recorded in the literature. Oxidation of the acetylated diphenylethylenes was performed a t 45-50' because of their insolubility in acetic acid a t 15'. The other procedures require no comment.
TABLE I DEGRADATION PRODUCTS OF LITHOCHOLIC ACID Compound
3-Acetoxy-bis-nor-cholanyldiphenylethylene 3-Acetoxypregnanyldiphenylethylene 1,l-Diphenyl-methyl-(3-acetoxy-etiocholany1)-ethylene Pregnanol-3 (a) -one-20 21-Benzalpregnanolone 3-Acetoxy-etio-cholanic acid etio-Lithocholic acid
M.p. OC.
[a126uai
Calcd.,
Formula
C
CssHsoO2 CsrHaOz
84.71 84.61
% H
p n d ,
%
H
9.36 9.22
158-160 150-152
+75.5 (in CHCls) f 140 * 3 O (in CHCla)
158-160 148-149 228-230 226-229 270-272
f 3 9 8 * 2 " (in CHClZ) C36H4602 84.65 9.08 84.73 9.31 10.76 78.85 10.74 +129 *.3' (in alcohol) C Z I H ~ ~ O79.19 Z 82.71 9.42 82.34 9.51 +181 * 3' (in alcohol) cZ&3802 f 8 6 . 4 * 3 ' (in alcohol) CzzHs404 72.87 9.46 72.70 9.33 CzoHazOa 74.96 10.07 74.51 10.06
Ozonolysis of l,l-diphenyl-methyl-(3-acetoxybile acids into materials suitable for the preparation of desoxycorticosterone and related sub- etio-cholany1)-ethylene' followed by hydrolysis gave pregnanol-3( a)-one-20.8 Further degradastances. The first step was the conversion of cholic and tion proceeded as with the corresponding ketone desoxycholic acids to lithocholic acid. This con- derived from desoxycholic acid. The benzal version has been accomplished by a number of ketone was isolated and its properties were found procedures3 but the over-all yields have not been to agree with those given by Marker and Wittles encouraging. By the action of benzoyl chloride who have recently used this procedure for the and pyridine on methyl desoxycholate a t 15' the preparation of etio-cholanic acids from the preg3-monobenzoate was obtained in good yield. Oxi- nanolones. The preparation of dehydro-etiodation t o methyl 3-benzoxy-12-ketocholanate lithocholic acid and 4-bromo-3-keto-etio-cholanic with chromic acid proceeded smoothly as did the acid by the usual methods offered no difficulties. subsequent preparation of the semicarbazone. Experimental Part The over-all yield of semicarbazone from methyl Methyl 3-Benzoxy-l2-hydroxycholanate.-A solution of desoxycholate was more than 80%. Reduction 32 g. (0.08 mole) of methyl desoxycholate in 150 cc. of dry of the semicarbazone by the WOE-Kishner benzene was cooled t o 5 ', 10 cc. of dry pyridine was added, method yielded an average of 60% of recrystal- and then 9.3 cc. (0.081 mole) of benzoyl chloride in 20 cc. lized lithocholic acid. (7) The Referee has pointed out that we have fallen into error in The degradation of lithocholic acid by the our previous paper' in naming the diphenylethylenes derived from and homologous acids. 3,12-Diacctoxy-bis-nor-cho2Wieland-Barbier method was performed as pre- desoxycholic cnyldiPhcnylcfhylcnc is correct for the compound called 3,la-diacetviously described for desoxycholic acid.4 Reindel oxy-nor-cholanyldipheeylethylene;3,1Z-diacelo+yprcgnanyldiphenyl(or 3,1Z-diaccloxy-tcr-nor-cholanyldiPhc~yle~h~lene) inand Niederlander5 and Sawlewicz and Reichstein6 ethylene stead of 3,12-diacetoxy-brs-nor-cholanyldiphenylethylene; and 1,1(1) Steiger and Reichstein, Hela. Chim. Acta, 20, 1164 (1937). diphcn~l-mcthyl-(3,12-diacctory-cfio-cho~a~yl)-ethylene instead of 3,(2) Serini, Logemann and Hildebrand, Bcr., 72, 391 (1939). 12-diacetoxy-tcr-nor-cholanyldiphenylethylene.The names previ(3) Recent papers are (a) Bergstrom and Haslewood, J . Chcm. ously applied included one carbon atom in both the ethylene group SOC. London, 540 (1939); (b) Dutcher and Wintersteiner, THIS and the cholanyl radical. JOURNAL, 61, 1992 (1939). ( 8 ) (a) Marker, Kamm and Jones, THISJOURNAL, 59, 1595 (1937); 14) Hoehn and Mason, THISJOURNAL, 60, 1493 (1938). (b) Marker, Kamm and Wittle, ibid., 59, 1841 (1937); (c) Butenandt (5) Reindel and Niederlander, Ber., 68, 1969 (1935). and Hildebrandt, U. S. Patent 2,156,275 (1939). (6) Sawlewicz and Reichstein, Hclu. Chim. Acta, 20, 949 (19371. (9) Marker and Wittle, THISJOURNAL, 61, 1329 (1939).
570
WILLARD
M. HOE"
AND
HAROLD L. MASON
Vol. 62
of dry benzene was added a t such a rate that no rise in for the preparation of the semicarbazone of methyl 3temperature occurred. The mixture was allowed to benzoxy-12-ketocholanate)was added to a solution of 70 come to room temperature and t o stand for three hours. g. of sodium in 700 cc. of methyl alcohol and heated in an The benzene solution was washed with three 100 cc. por- autoclave at 174 * 5" for six hours. After solution tions of 0.5 N hydrochloric acid, then with 0.2 N sodium of the reaction products in warm water the methyl alhydroxide and finally with water. After removal of water cohol was removed in a vacuum. The aqueous fraction with anhydrous sodium sulfate, the benzene was distilled was warmed until all solids were dissolved and was then in a vacuum. The glassy residue was dissolved by warm- poured with stirring into an excess of dilute hydrochloric ing with 200 cc. of dry ether. The etherate of methyl acid. The precipitated acid, which was often somewhat 3-benzoxy-12-hydroxycholanate crystallized from the gummy, was suspended in 0.8 N sodium hydroxide solusolution on standing. It was air-dried and then melted tion and heated to 40-50 O on the water-bath. The sodium a t 78-80" with frothing; yield, 8749%. lithocholate which separated under these conditions was Anal. Calcd. for C32H4606.1/zCpH100: C, 74.55; H, filtered out, resuspended in 1 N sodium hydroxide and again isolated. It was then dissolved in hot water and 9.39. Found: C, 74.35; H, 9.36. When two molar equivalents of benzoyl chloride and the solution was poured into a slight excess of dilute hypyridine were used, the dibenzoate of methyl desoxy- drochloric acid. The lithocholic acid was filtered off and washed well with water. Dried a t 110" and crystallized cholate was obtained, m. p. 145-146". Methyl 3-Benzoxy-l2-ketocholanate.-A solution of 44 from dry acetone, the yield was 25.9 g. (62.5%) of lithog. (0.08 mole) of methyl 3-benzoxy-12-hydroxycholanate cholic acid (m. p. 183.5-185'). Dehydro-etio-lithocholic Acid.-A solution of 390 mg. etherate in 70 cc. of acetic acid was evaporated in a vac(0.00124 mole) of etio-lithocholic acid in 20 cc. of glacial uum to remove the ether, and the residue was dissolved in 400 cc. of glacial acetic acid. To this solution, cooled to acetic acid was cooled to 15' and 100 mg. of chromic acid 15", was added 8 g. of chromic acid in 100 cc. of 90% in 1 cc. of 90% acetic acid was added. After one hour the acetic acid. The mixture stood overnight in the refrigera- excess of chromic acid was reduced with alcohol. The tor and was worked up in the usual manner for the neutral solvents were removed in a vacuum and the residue was product. This substance usually was not isolated. It extracted with 200 cc. of ether and 100 cc. of water. The ether layer was separated, washed with water until colorcan be crystallized in the icebox from a large volume of less and dried over anhydrous sodium sulfate. The acid, absolute alcohol and then melts a t 94-95'. Semicarbazone.-The crude keto ester was dissolved in after crystallization from acetone, weighed 330 mg. (84%). Crystallization from benzene gave a product which melted 250 cc. of alcohol and a solution of 19 g. (0.17 mole) of at 246-245'. Marker and Wittles report a melting point semicarbazide hydrochloride and 15 g. of sodium acetate in 100 cc. of 80% alcohol was added. This mixture was of 246-249' but no analytical figures are given. Anal. Calcd. for CloHs~Oa:C, 75.43; H, 9.49. Found: refluxed for fourteen hours. The bulk of the semicarbazone separated during the refluxing and was removed by C, 75.46; H, 9.70. filtration of the hot solution. A second crop, obtained by 4-Bromo-dehydro-etio-lithocholic Acid.-To a solution cooling, was purified by boiling with water. The tota; of 784 mg. (0.00246 mole) of dehydro-ctio-lithocholicacid yield, after drying a t 110" for eight hours, was 43 g. (95% in 30 cc. of glacial acetic acid a t 18" was added 6.3 cc. of based on the etherate). It melted a t 160-162" after re- 0.82 N bromine in glacial acetic acid solution. After standing ten minutes the solvents were removed in a vaccrystallization from alcohol. Lithocholic Acid (I).-A mixture containing 45 g. of so- uum. The residue was taken .up in ethyl acetate from dium and 43 g. (0.076 mole) of the semicarbazone in 400 cc. which crystallized 620 mg. (63.5%); m. p. 190-192'. of absolute alcohol was heated in an autoclave a t about Anal. Calcd. for CZoH2008Br: C, 60.45; H, 7.19. 200' for seven hours. The reaction product was dissolved Found: C, 60.30; H , 7.40. in hot water and acidified to congo red with dilute hydrochloric acid. The precipitated acid was washed on the Summary filter with hot water, dried and crystallized from acetone. The yield of lithocholic acid (m. p. 183-185'; [ C Y ] ~ ~ D Lithocholic acid has been prepared from cholic f34'; [ C Y I ~+39") ~ M ~was ~ ~18.4 g. (64.6%). and desoxycholic acids with an over-all yield of Anal. Calcd. for CzrH4~Os: C, 76.55; H , 10.71. 50%. Some of the intermediate compounds of Found: C, 76.36; H, 10.68. the Wolff-Kishner degradation of lithocholic Lithocholic Acid (2).--Seventy grams (0.110 mole) of acid have been described. the disemicarbazone of methyl 3-benzoxy-7,12-diketocholanate (prepared by procedures similar t o those used ROCHESTER, MINNESOTA RECEIVEDDECEMBER 18,1939
